P1367: REFINED PHENOTYPE OF SENESCENT T CELLS UNVEILS HIGH ANTI-TUMOR ACTIVITY DESPITE IMPAIRED PROLIFERATION AND DISTURBED METABOLIC ACTIVITY: NEW IMPLICATIONS FOR CANCER IMMUNOTHERAPY
Authors: Kevin Legscha, Abdulkadir Yilmaz, Lea Märker, Edite Antunes, Catherine Wölfel, Borhane Guezguez, Stefan Porubsky, Matthias Theobald, Hakim Echchannaoui
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Despite the success of T cell-based cancer immunotherapy, a large fraction of patients still does not benefit from this treatment approach. T cell dysfunction associated with old age, or the hostile tumor microenvironment often leads to impaired anti-tumor responses in these patients. Exhaustion is one well-described mechanism driving T cells towards a non-responsive state. However, few studies suggest that in some cases, senescence rather than exhaustion is the main driver of poor anti-tumor responses. Senescence in T cells shares some characteristics with exhaustion but is thought to be induced by different mechanisms and remains less well understood. Aims: This study aims to characterize the mechanisms of senescence-induced T cell dysfunction and to test their cytotoxic potential for adoptive T cell therapy for cancer. Improving the understanding of senescence in T cells and its underlying mechanisms should help to identify methods to revert senescence-induced cell cycle arrest and utilize the cytolytic potential of these effector cells. Methods: In this study, we characterized CD8+ T cells from healthy, but aged donors extensively regarding their cellular phenotype and differentiation status by flow cytometry. We then correlated these findings with the functional features (proliferation, cytotoxicity) and metabolic activity (metabolic flux assays, microscopy). To test the cytotoxic potential of identified senescent T cells, we introduced a tumor antigen-specific T cell receptor (TCR) by RNA-electroporation into different T cell populations and tested their cytolytic activity. In ongoing experiments, we additionally perform RNA sequencing on a single cell level to identify pathways that regulate and maintain senescence in these cells. Results: Using this multi-analysis approach, we could identify a distinct and highly differentiated subpopulation of non-proliferative T cells within the “conventional” senescent T cell population by a combination of surface markers (e.g., CD28, CD57 and CD45RA). Furthermore, this senescent state was associated with an impaired mitochondrial function as shown by reduced respiration and membrane potential. Altered mitochondrial function also correlated with morphological changes of the mitochondria as demonstrated by electron microscopy. Again, only the combination of multiple “senescent marker” was sufficient to identify the senescent population with disturbed mitochondrial function. In turn, senescence-inducing treatments caused similar phenotypes with altered mitochondrial activity. Additionally, inhibition of mitochondrial respiration promoted the onset of senescent in healthy T cells. On a functional level, the senescent T cells showed a strong cytotoxic potential indicated by high production of effector molecules and degranulation. However, a direct proof of potent anti-tumor cytotoxicity of this T cell population is still missing. Therefore, we performed long-term tumor cytolytic assays, in which the senescent T cells elicited a strong anti-tumor response despite being non-proliferative and prone to apoptosis. Summary/Conclusion: These results highlight the potential of senescent effector T cell populations in cancer immunotherapy. This may help to enhance our understanding of senescence in T cells and might identify strategies to revert senescence-induced dysfunction and improve T cell-based cancer immunotherapies. Keywords: Mitochondria, Senescence, Adoptive immunotherapy, Antigen-specific T cells